Skeletal muscle hexokinase: regulation in mammalian hibernation Khalil Abnous Æ Kenneth B. Storey Received: 20 March 2008 / Accepted: 3 July 2008 / Published online: 20 July 2008 Ó Springer Science+Business Media, LLC. 2008 Abstract Skeletal muscle hexokinase (HK) from Rich- ardson’s ground squirrels was analyzed to determine how the enzyme is regulated during hibernation, a state of cold torpor. The HK II isozyme dominated in muscle and *15% of total HK was bound to the insoluble fraction. HK maximum activity was 33% lower in hibernator muscle and the enzyme showed a significantly higher K m ATP (by 80%) and a lower K i for glucose-6-P (by 40%) than euthermic HK (assayed at 22°C). However, 5°C assay significantly reduced K m glucose of hibernator HK. Stim- ulation of AMP-dependent protein kinase (AMPK) in hibernator extracts elevated the HK activity and reduced K m ATP, but did not affect euthermic HK. Stimulation of protein phosphatases significantly lowered the HK activity in both situations. AMPK-dependent phosphorylation was confirmed by immunopreciptiation of 32 P-labeled HK. DEAE-Sephadex ion exchange chromatography revealed two peaks of HK in hibernator muscle extracts (low and high phosphate forms), whereas only a single peak of phospho-HK was present in euthermic muscle. We con- clude that differential control of muscle HK in euthermic versus hibernating states is derived from two main regu- latory influences, reversible protein phosphorylation and temperature effects on kinetic properties. Keywords Glucose catabolism Á Torpor Á Reversible phosphorylation Á Temperature effects Á Spermophilus richardsonii Introduction Glucose is a key metabolite for living organisms. The first step in its metabolism is phosphorylation by the enzyme hexokinase (HK; E.C. 2.7.1.1) to form glucose-6-phosphate (G6P) that can then enter a variety of pathways. These include glycolysis to generate ATP, the pentose phosphate pathway to produce NADPH and sugar phosphates, and the synthesis of glycogen as a storage fuel for future use [1]. HK catalyzes the reaction: glucose + ATP ? G6P + ADP. In mammals, there are four isozymes (HK I-IV) that differ in subcellular and tissue distribution [2, 3]. HK IV is also known as glucokinase; it is liver-specific with a high K m glucose. The other three isozymes have molecular weights of *100 kD and low K m glucose values; they can be separated by ion exchange chromatography and isoelectric focusing [2, 3]. Differential transcriptional regulation of HK isozymes may permit their selective expression in particular tissues and HK involvement in various metabolic applications [4]. The isozymes differ in their affinity for substrates (ATP, glucose) and in their inhibition by phosphate and G6P [1]. The HKI isozyme has a hydrophobic N-terminal sequence that allows it to bind to the voltage-dependent anion channel (VDAC) on the outer membrane of mitochondria where it can make preferential use of ATP that is provided from oxidative phosphorylation [1, 5]. HKII also has this ability whereas some HKIII is associated the nuclear membrane although both of these isozymes are also found free in the cytoplasm. Hibernation is a critical winter survival strategy for many small mammals. In order to endure unfavorable environmental conditions, animals strongly suppress their energy-consuming physiological functions and enter a state of cold torpor. During hibernation in ground squirrels, body temperature falls to near 0°C, heart beat is reduced from its K. Abnous Á K. B. Storey (&) Institute of Biochemistry and Department of Chemistry, Carleton University, 1125 Colonel By Drive, Ottawa, ON, Canada K1S 5B6 e-mail: kenneth_storey@carleton.ca 123 Mol Cell Biochem (2008) 319:41–50 DOI 10.1007/s11010-008-9875-5