Disruption of the allosteric phosphorylase a regulation of the hepatic glycogen-targeted protein phosphatase 1 improves glucose tolerance in vivo.

Type 2 diabetes is characterised by elevated blood glucose concentrations, which potentially could be normalised by stimulation of hepatic glycogen synthesis. Under glycogenolytic conditions, the interaction of hepatic glycogen-associated protein phosphatase-1 (PP1-G(L)) with glycogen phosphorylase a is believed to inhibit the dephosphorylation and activation of glycogen synthase (GS) by the PP1-G(L) complex, suppressing glycogen synthesis. Consequently, the interaction of G(L) with phosphorylase a has emerged as an attractive anti-diabetic target, pharmacological disruption of which could provide a novel mechanism to lower blood glucose levels by increasing hepatic glycogen synthesis. Here we report for the first time the in vivo consequences of disrupting the G(L)-phosphorylase a interaction, using a mouse model containing a Tyr284Phe substitution in the phosphorylase a-binding region of the G(L) protein. The resulting G(L)(Y284F/Y284F) mice display hepatic PP1-G(L) activity that is no longer sensitive to allosteric inhibition by phosphorylase a, resulting in increased GS activity under glycogenolytic conditions, demonstrating that regulation of G(L) by phosphorylase a operates in vivo. G(L)(Y284F/Y284F) and G(L)(Y284F/+) mice display improved glucose tolerance compared with G(L)(+/+) littermates, without significant accumulation of hepatic glycogen. The data provide the first in vivo evidence in support of targeting the G(L)-phosphorylase a interaction for treatment of hyperglycaemia. During prolonged fasting the G(L)(Y284F/Y284F) mice lose more body weight and display decreased blood glucose levels in comparison with their G(L)(+/+) littermates. These results suggest that, during periods of food deprivation, the phosphorylase a regulation of G(L) may prevent futile glucose-glycogen cycling, preserving energy and thus providing a selective biological advantage that may explain the observed conservation of the allosteric regulation of PP1-G(L) by phosphorylase a in mammals.

[1]  L. Agius.  Glucokinase and molecular aspects of liver glycogen metabolism. , 2008, The Biochemical journal.

[2]  S. Wild,et al.  Global prevalence of diabetes: estimates for the year 2000 and projections for 2030. , 2004, Diabetes care.

[3]  R. Gomis,et al.  Control of glycogen deposition , 2003, FEBS letters.

[4]  P. Cohen,et al.  Phosphorylase a is an allosteric inhibitor of the glycogen and microsomal forms of rat hepatic protein phosphatase‐1 , 1986, FEBS letters.

[5]  J. Timmons,et al.  The Experimental Type 2 Diabetes Therapy Glycogen Phosphorylase Inhibition Can Impair Aerobic Muscle Function During Prolonged Contraction , 2006, Diabetes.

[6]  G. Browne,et al.  Assay of protein phosphatase 1 complexes. , 2003, Methods in enzymology.

[7]  B. Doble,et al.  Glycogen synthase kinase-3 in insulin and Wnt signalling: a double-edged sword? , 2004, Biochemical Society transactions.

[8]  P. Roach,et al.  Glycogen and its metabolism. , 2002, Current molecular medicine.

[9]  Philip Cohen,et al.  The twentieth century struggle to decipher insulin signalling , 2006, Nature Reviews Molecular Cell Biology.

[10]  M. Sales,et al.  Human Skeletal Muscle Expresses a Glycogen-Targeting Subunit of PP1 That Is Identical to the Insulin-Sensitive Glycogen-Targeting Subunit GL of Liver , 2002 .

[11]  S. Kimura,et al.  Identification and expression of alternative splice variants of the mouse Ppp1r3b gene in lung epithelial cells. , 2006, Biochemical and biophysical research communications.

[12]  A. Depaoli-Roach,et al.  Mice with elevated muscle glycogen stores do not have improved exercise performance. , 2005, Biochemical and biophysical research communications.

[13]  R. Jope,et al.  The glamour and gloom of glycogen synthase kinase-3. , 2004, Trends in biochemical sciences.

[14]  A. Pautsch,et al.  Molecular Recognition of the Protein Phosphatase 1 Glycogen Targeting Subunit by Glycogen Phosphorylase* , 2008, Journal of Biological Chemistry.

[15]  Hans Clevers,et al.  Caught up in a Wnt storm: Wnt signaling in cancer. , 2003, Biochimica et biophysica acta.

[16]  P. Cohen,et al.  Amino acid sequence and expression of the hepatic glycogen‐binding (GL‐subunit of protein phosphatase‐1 , 1995, FEBS letters.

[17]  D. Hardie.  Protein phosphorylation: a practical approach , 1993 .

[18]  Ian R. Kelsall,et al.  Disruption of the striated muscle glycogen-targeting subunit of protein phosphatase 1: influence of the genetic background. , 2008, Journal of molecular endocrinology.

[19]  J. H. Roe,et al.  Determination of glycogen with the anthrone reagent. , 1966, Analytical biochemistry.

[20]  J. Treadway,et al.  Glycogen phosphorylase inhibitors for treatment of type 2 diabetes mellitus , 2001, Expert opinion on investigational drugs.

[21]  C G Armstrong,et al.  Identification of the separate domains in the hepatic glycogen-targeting subunit of protein phosphatase 1 that interact with phosphorylase a, glycogen and protein phosphatase 1. , 1998, The Biochemical journal.

[22]  J. A. Thomas,et al.  A rapid filter paper assay for UDPglucose-glycogen glucosyltransferase, including an improved biosynthesis of UDP-14C-glucose. , 1968, Analytical biochemistry.

[23]  M. Bollen,et al.  Loss of the hepatic glycogen-binding subunit (GL) of protein phosphatase 1 underlies deficient glycogen synthesis in insulin-dependent diabetic rats and in adrenalectomized starved rats. , 1998, The Biochemical journal.

[24]  W Stalmans,et al.  High-affinity binding of glycogen-synthase phosphatase to glycogen particles in the liver. Role of glycogen in the inhibition of synthase phosphatase by phosphorylase a. , 1987, The Biochemical journal.

[25]  P. Cohen.  [26] Phosphorylase kinase from rabbit skeletal muscle , 1983 .

[26]  P. Cohen,et al.  Purification of the hepatic glycogen‐associated form of protein phosphatase‐1 by microcystin‐Sepharose affinity chromatography , 1995, FEBS letters.

[27]  L. Agius,et al.  Diverse effects of two allosteric inhibitors on the phosphorylation state of glycogen phosphorylase in hepatocytes. , 2002, The Biochemical journal.

[28]  S. Kauschke,et al.  Inhibition of the interaction between protein phosphatase 1 glycogen-targeting subunit and glycogen phosphorylase increases glycogen synthesis in primary rat hepatocytes. , 2008, The Biochemical journal.

[29]  P. Cohen,et al.  NIMA-related kinase 2 (Nek2), a cell-cycle-regulated protein kinase localized to centrosomes, is complexed to protein phosphatase 1. , 2000, The Biochemical journal.

[30]  P. Cohen,et al.  The skeletal muscle-specific glycogen-targeted protein phosphatase 1 plays a major role in the regulation of glycogen metabolism by adrenaline in vivo. , 2007, Cellular signalling.

[31]  L. Agius.  New hepatic targets for glycaemic control in diabetes. , 2007, Best practice & research. Clinical endocrinology & metabolism.

[32]  H. Hers,et al.  The influence of inorganic phosphate, adenosine triphosphate and glucose 6-phosphate on the activity of liver glycogen synthetase. , 1968, European journal of biochemistry.

[33]  M. Bollen,et al.  Specific features of glycogen metabolism in the liver. , 1998, The Biochemical journal.

[34]  H. Berman,et al.  Distinctive Regulatory and Metabolic Properties of Glycogen-targeting Subunits of Protein Phosphatase-1 (PTG, GL, GM/RGl) Expressed in Hepatocytes* , 2000, The Journal of Biological Chemistry.

[35]  B. Doble,et al.  Glycogen synthase kinase 3alpha-specific regulation of murine hepatic glycogen metabolism. , 2007, Cell metabolism.

[36]  H. Hers,et al.  The stimulation of liver phosphorylase b by AMP, fluoride and sulfate. A technical note on the specific determination of the a and b forms of liver glycogen phosphorylase. , 1975, European journal of biochemistry.

[37]  J. Guinovart,et al.  Hepatic Glycogen Synthesis Is Highly Sensitive to Phosphorylase Activity , 2001, The Journal of Biological Chemistry.

[38]  P. Cohen,et al.  The purification and properties of rabbit skeletal muscle glycogen synthase. , 1976, European journal of biochemistry.

[39]  D. Graves,et al.  Rabbit-liver phosphorylase: improvement of the purification procedure and assay of the inactive b form. , 1982, Analytical biochemistry.

[40]  R. DeFronzo.  Pharmacologic Therapy for Type 2 Diabetes Mellitus , 1999, Annals of Internal Medicine.

[41]  C. Lerin,et al.  Expression and glycogenic effect of glycogen-targeting protein phosphatase 1 regulatory subunit GL in cultured human muscle. , 2007, The Biochemical journal.

[42]  P. Cohen,et al.  Glycogen synthase kinase-3 from rabbit skeletal muscle. , 2005, Methods in enzymology.

[43]  J. Treadway,et al.  The hepatic PP1 glycogen‐targeting subunit interaction with phosphorylase a can be blocked by C‐terminal tyrosine deletion or an indole drug , 2007, FEBS letters.

[44]  G. Seghieri,et al.  Are the available experimental models of type 2 diabetes appropriate for a gender perspective? , 2008, Pharmacological research.

[45]  D. Graves,et al.  The effect of solvents on nucleotide regulation of glycogen phosphorylase. , 1979, The Journal of biological chemistry.

[46]  G. Browne,et al.  The level of the glycogen targetting regulatory subunit R5 of protein phosphatase 1 is decreased in the livers of insulin-dependent diabetic rats and starved rats. , 2001, The Biochemical journal.

[47]  W. Stalmans,et al.  The inhibitory effect of phosphorylase a on the activation of glycogen synthase depends on the type of synthase phosphatase. , 1983, The Biochemical journal.