title: Suppression of 5'-nucleotidase enzymes promotes AMP-activated protein kinase (AMPK) phosphorylation and metabolism in human and mouse skeletal muscle.
abstract: The 5'-nucleotidase (NT5) family of enzyme dephosphorylates non-cyclic nucleoside monophosphates to produce nucleosides and inorganic phosphates. We hypothesized that gene silencing of NT5 enzymes to increase the intracellular availability of AMP would increase AMP-activated protein kinase (AMPK) activity and metabolism. We determined the role of cytosolic NT5 in metabolic responses linked to the development of insulin resistance in obesity and type 2 diabetes. Using siRNA to silence NT5C2 expression in cultured human myotubes, we observed a 2-fold increase in the AMP/ATP ratio, a 2.4-fold increase in AMPK phosphorylation (Thr(172)), and a 2.8-fold increase in acetyl-CoA carboxylase phosphorylation (Ser(79)) (p < 0.05). siRNA silencing of NT5C2 expression increased palmitate oxidation by 2-fold in the absence and by 8-fold in the presence of 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside. This was paralleled by an increase in glucose transport and a decrease in glucose oxidation, incorporation into glycogen, and lactate release from NT5C2-depleted myotubes. Gene silencing of NT5C1A by shRNA injection and electroporation in mouse tibialis anterior muscle reduced protein content (60%; p < 0.05) and increased phosphorylation of AMPK (60%; p < 0.05) and acetyl-CoA carboxylase (50%; p < 0.05) and glucose uptake (20%; p < 0.05). Endogenous expression of NT5C enzymes inhibited basal lipid oxidation and glucose transport in skeletal muscle. Reduction of 5'-nucleotidase expression or activity may promote metabolic flexibility in type 2 diabetes.
authors.affiliation: Department of Molecular Medicine and Surgery and Physiology and Pharmacology, Karolinska Institutet, S-171 77 Stockholm, Sweden.
authors.forename: Sameer S
authors.initials: SS
authors.lastname: Kulkarni
authors.forename: Håkan K R
authors.initials: HK
authors.lastname: Karlsson
authors.forename: Ferenc
authors.initials: F
authors.lastname: Szekeres
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Chibalin
authors.forename: Anna
authors.initials: A
authors.lastname: Krook
authors.forename: Juleen R
authors.initials: JR
authors.lastname: Zierath
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000378:metabolism*; D000818:Animals; D003921:Diabetes Mellitus, Experimental; D005786:Gene Expression Regulation; D020868:Gene Silencing; D005947:Glucose / Q000737:chemistry; D006003:Glycogen / Q000737:chemistry; D006801:Humans; D008055:Lipids / Q000737:chemistry; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018482:Muscle, Skeletal / Q000378:metabolism*; D019308:Palmitic Acid / Q000378:metabolism; D010766:Phosphorylation
chemical_list: D008055:Lipids; D019308:Palmitic Acid; D006003:Glycogen; D055372:AMP-Activated Protein Kinases; D005947:Glucose