SUMMARY Glycogen is a branched polymer of glucose that serves as an energy store. Phosphate, a trace constituent of glycogen, has profound effects on glycogen structure and phosphate hyperaccumulation is linked to Lafora disease, a fatal progressive myoclonus epilepsy that can be caused by mutations of laforin, a glycogen phosphatase. However, little is known about the metabolism of glycogen phosphate. We demonstrate here that the biosynthetic enzyme glycogen synthase, which normally adds glucose residues to glycogen, is capable of incorporating the β-phosphate of its substrate UDP-glucose at a rate of one phosphate per approximately 10,000 glucoses, in what may be considered a catalytic error. We show that the phosphate in glycogen is present as C2 and C3 phosphomonoesters. Since hyperphosphorylation of glycogen causes Lafora disease, phosphate removal by laforin may thus be considered a repair or damage control mechanism.

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Background: Lafora disease is characterized by abnormal, hyperphosphorylated glycogen. Results: 20% of the total phosphate is present as a C6 phosphomonoester of glucose residues; this proportion is unchanged in glycogen from mouse models of Lafora disease. Conclusion: C6 phosphate is not the dominant phosphomonoester. Significance: C2, C3, or C6 phosphate could all contribute to aberrant glycogen structure.

Glycogen synthase is a rate-limiting enzyme in the biosynthesis of glycogen and has an essential role in glucose homeostasis. The three-dimensional structures of yeast glycogen synthase (Gsy2p) complexed with maltooctaose identified four conserved maltodextrin-binding sites distributed across the surface of the enzyme. Site-1 is positioned on the N-terminal domain, site-2 and site-3 are present on the C-terminal domain, and site-4 is located in an interdomain cleft adjacent to the active site. Mutation of these surface sites decreased glycogen binding and catalytic efficiency toward glycogen. Mutations within site-1 and site-2 reduced the V max /S 0.5 for glycogen by 40-and 70-fold, respectively. Combined mutation of site-1 and site-2 decreased the V max /S 0.5 for glycogen by >3000-fold. Consistent with the in vitro data, glycogen accumulation in glycogen synthase-deficient yeast cells (⌬gsy1-gsy2) transformed with the site-1, site-2, combined site-1/site-2, or site-4 mutant form of Gsy2p was decreased by up to 40-fold. In contrast to the glycogen results, the ability to utilize maltooctaose as an in vitro substrate was unaffected in the site-2 mutant, moderately affected in the site-1 mutant, and almost completely abolished in the site-4 mutant. These data show that the ability to utilize maltooctaose as a substrate can be independent of the ability to utilize glycogen. Our data support the hypothesis that site-1 and site-2 provide a "toehold mechanism," keeping glycogen synthase tightly associated with the glycogen particle, whereas site-4 is more closely associated with positioning of the nonreducing end during catalysis.Glycogen synthase was the first reported intracellular target of insulin, and the enzyme catalyzes the linear polymerization of glucose residues from activated sugar donor molecules to the nonreducing end of the glycogen chain. Recent structural studies have shown that the enzyme folds into two Rossmann foldlike domains, with a deep cleft in between harboring the active site (1-3). Although the basic fold is conserved between the prokaryotic, archaeal, and eukaryotic enzymes, there are multiple sequence insertions in the eukaryotic enzymes. The largest of these insertions (a long coiled-coil insert in the C-terminal domain) gives rise to their unique tetrameric arrangement as well as the structural plasticity necessary for the complex regulation of glycogen synthase activity in eukaryotes (3). Furthermore, a conserved arginine cluster present in the C-terminal region of the eukaryotic enzymes mediates the sensitivity to inhibition by phosphorylation and activation by glucose 6-phosphate (4, 5). In our recent structural studies, we demonstrated that the middle two arginine residues 3 are necessary and sufficient to confer regulation by glucose 6-phosphate and that the first three arginine residues (Arg-580, Arg-581, and Arg-583) are required for full regulatory response to phosphorylation (3).The yeast Saccharomyces cerevisiae possesses two genes encoding glycogen synthase, GSY1 and GSY2, wh...

Significance Glycogen is a branched glucose polymer found in most animals, fungi, bacteria, and archaea as an osmotically neutral means of energy storage. Glycogen also contains minor amounts of phosphate which can be removed by a dual specificity phosphatase, laforin. Accumulation of phosphate results in highly insoluble glycogen deposits and underlies Lafora disease, a devastating form of myoclonus epilepsy. In this paper, we present structural and kinetic data that support a plausible mechanism by which phosphate is directly incorporated into glycogen by glycogen synthase.

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β-cell death is regarded as a major event driving loss of insulin secretion and hyperglycemia in both type 1 and type 2 diabetes mellitus. In this review, we explore past, present, and potential future advances in our understanding of the mechanisms that promote β-cell death in diabetes, with a focus on the primary literature. We first review discoveries of insulin insufficiency, β-cell loss, and β-cell death in human diabetes. We discuss findings in humans and mouse models of diabetes related to autoimmune-associated β-cell loss and the roles of autoreactive T cells, B cells, and the β cell itself in this process. We review discoveries of the molecular mechanisms that underlie β-cell death-inducing stimuli, including proinflammatory cytokines, islet amyloid formation, ER stress, oxidative stress, glucotoxicity, and lipotoxicity. Finally, we explore recent perspectives on β-cell death in diabetes, including: (1) the role of the β cell in its own demise, (2) methods and terminology for identifying diverse mechanisms of β-cell death, and (3) whether non-canonical forms of β-cell death, such as regulated necrosis, contribute to islet inflammation and β-cell loss in diabetes. We believe new perspectives on the mechanisms of β-cell death in diabetes will provide a better understanding of this pathological process and may lead to new therapeutic strategies to protect β cells in the setting of diabetes.