There is growing evidence that glycogen targeting subunits of protein phosphatase-1 play a critical role in regulation of glycogen metabolism. In the current study, we have investigated the effects of adenovirus-mediated overexpression of a specific glycogen targeting subunit known as protein targeting to glycogen (PTG) in cultured human muscle cells. PTG was overexpressed both in muscle cells cultured at high glucose (glycogen replete) or in cells incubated for 18 h in the absence of glucose and then incubated in high glucose (glycogen re-synthesizing). In both glycogen replete and glycogen resynthesizing cells, PTG overexpression caused glycogen to be synthesized at a linear rate 1-5 days after viral treatment, while in cells treated with a virus lacking a cDNA insert (control virus), glycogen content reached a plateau at day 1 with no further increase. In the glycogen replete PTG overexpressing cells, glycogen content was 20 times that in controls at day 5. Furthermore, in cells undergoing glycogen resynthesis, PTG overexpression caused a doubling of the initial rate of glycogen synthesis over the first 24 h relative to cells treated with control virus. In both sets of experiments, the effects of PTG on glycogen synthesis were correlated with a 2-3-fold increase in glycogen synthase activity state, with no changes in glycogen phosphorylase activity. The alterations in glycogen synthase activity were not accompanied by changes in the intracellular concentration of glucose 6-phosphate. We conclude that PTG overexpression activates glycogen synthesis in a glucose 6-phosphate-independent manner in human muscle cells while overriding glycogen-mediated inhibition. Our findings suggest that modulation of PTG expression in muscle may be a mechanism for enhancing muscle glucose disposal and improving glucose tolerance in diabetes.Glycogen metabolism is regulated in part by a balance between glycogen synthase (GS) 1 and glycogen phosphorylase (GP) activities. Both enzymes are known to be modified by phosphorylation-dephosphorylation reactions. Dephosphorylation of GS causes its activation, while GP becomes inactivated. Dephosphorylation of glycogen metabolizing enzymes has been mainly attributed to protein phosphatase 1 (PP1) activity (1). In support of this notion, PP1 is known to bind to the glycogen particle in muscle, and has also been shown to catalyze dephosphorylation of GS, GP, and phosphorylase kinase when assayed in vitro (2). However, less is known about the mechanisms that control the specific action of PP1 on glycogen metabolizing enzymes in the intact cell.It is increasingly appreciated that the activity of PP1 is affected by proteins that bind to the enzyme and target it to specific intracellular sites. With regard to glycogen metabolism, a family of glycogen targeting subunits of PP1 have recently emerged. These proteins include G M or R Gl (3) which is expressed in skeletal muscle, G L (4), expressed mainly in the liver, and PPP1R5 or protein targeting to glycogen (PTG) (5, 6) and PPP1R6 (7), which are...
G M , the muscle-specific glycogen-targeting subunit of protein phosphatase 1 (PP1) targeted to the sarcoplasmic reticulum, was proposed to regulate recovery of glycogen in exercised muscle, whereas mutation truncation of its COOH-terminal domain is known to be associated with type 2 diabetes. Here, we demonstrate differential effects of G M overexpression in human muscle cells according to glycogen concentration. Adenovirus-mediated delivery of G M slightly activated glycogen synthase (GS) and inactivated glycogen phosphorylase (GP) in glycogen-replete cells, causing an overaccumulation of glycogen and impairment of glycogenolysis after glucose deprivation. Differently, in glycogendepleted cells, G M strongly increased GS activation with no further enhancement of early glycogen resynthesis and without affecting GP. Effects of G M on GS and GP were abrogated by treatment with dibutyryl cyclic AMP. Expression of a COOH-terminal deleted-mutant (G M ⌬C), lacking the membrane binding sequence to sarcoplasmic reticulum, failed to activate GS in glycogen-depleted cells, while behaving similar to native G M in glycogen-replete cells. This is explained by loss of stability of the G M ⌬C protein following glycogen-depletion. In summary, G M promotes glycogen storage and inversely regulates GS and GP activities, while, specifically, synthase phosphatase activity of G M -PP1 is inhibited by glycogen. The conditional loss of function of the COOH-terminal deleted G M construct may help to explain the reported association of truncation mutation of G M with insulin resistance in human subjects. Diabetes
Pharmacological inhibition of liver GP (glycogen phosphorylase), which is currently being studied as a treatment for Type II (non-insulin-dependent) diabetes, may affect muscle glycogen metabolism. In the present study, we analysed the effects of the GP inhibitor CP-91149 on non-engineered or GP-overexpressing cultured human muscle cells. We found that CP-91149 treatment decreased muscle GP activity by (1) converting the phosphorylated AMP-independent a form into the dephosphorylated AMP-dependent b form and (2) inhibiting GP a activity and AMP-mediated GP b activation. Dephosphorylation of GP was exerted, irrespective of incubation of the cells with glucose, whereas inhibition of its activity was synergic with glucose. As expected, CP-91149 impaired the glycogenolysis induced by glucose deprivation. CP-91149 also promoted the dephosphorylation and activation of GS (glycogen synthase) in non-engineered or GP-overexpressing cultured human muscle cells, but exclusively in glucose-deprived cells. However, this inhibitor did not activate GS in glucose-deprived but glycogen-replete cells overexpressing PTG (protein targeting to glycogen), thus suggesting that glycogen inhibits the CP-91149-mediated activation of GS. Consistently, CP-91149 promoted glycogen resynthesis, but not its overaccumulation. Hence, treatment with CP-91149 impairs muscle glycogen breakdown, but enhances its recovery, which may be useful for the treatment of Type II (insulin-dependent) diabetes.
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