Inhibition of glucokinase translocation by AMP-activated protein kinase is associated with phosphorylation of both GKRP and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

Abstract

The rate of glucose phosphorylation in hepatocytes is determined by the subcellular location of glucokinase and by its association with its regulatory protein (GKRP) in the nucleus. Elevated glucose concentrations and precursors of fructose 1-phosphate (e.g., sorbitol) cause dissociation of glucokinase from GKRP and translocation to the cytoplasm. In this study, we investigated the counter-regulation of substrate-induced translocation by AICAR (5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside), which is metabolized by hepatocytes to an AMP analog, and causes activation of AMP-activated protein kinase (AMPK) and depletion of ATP. During incubation of hepatocytes with 25 mM glucose, AICAR concentrations below 200 microM activated AMPK without depleting ATP and inhibited glucose phosphorylation and glucokinase translocation with half-maximal effect at 100-140 microM. Glucose phosphorylation and glucokinase translocation correlated inversely with AMPK activity. AICAR also counteracted translocation induced by a glucokinase activator and partially counteracted translocation by sorbitol. However, AICAR did not block the reversal of translocation (from cytoplasm to nucleus) after substrate withdrawal. Inhibition of glucose-induced translocation by AICAR was greater than inhibition by glucagon and was associated with phosphorylation of both GKRP and the cytoplasmic glucokinase binding protein, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK2) on ser-32. Expression of a kinase-active PFK2 variant lacking ser-32 partially reversed the inhibition of translocation by AICAR. Phosphorylation of GKRP by AMPK partially counteracted its inhibitory effect on glucokinase activity, suggesting altered interaction of glucokinase and GKRP. In summary, mechanisms downstream of AMPK activation, involving phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and GKRP are involved in the ATP-independent inhibition of glucose-induced glucokinase translocation by AICAR in hepatocytes.

5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) Effect on Glucose Production, but Not Energy Metabolism, Is Independent of Hepatic AMPK in Vivo

“…AICAR delivery into the portal vein renders the liver insensitive to net hepatic glucose uptake, even during hyperglycemia and hyperinsulinemia (52). In vitro work confirms that AICAR reduces glucokinase translocation and glycolysis in hepatocytes (55), in AMPKdependent (56) and -independent (57) conditions. Our AICAR delivery rate was selected to permit a reasonable physiological comparison between our results and earlier AICAR-euglycemic clamps in rodents (24,25).…”

“…Our AICAR delivery rate was selected to permit a reasonable physiological comparison between our results and earlier AICAR-euglycemic clamps in rodents (24,25). The resulting dose was sufficient to perturb hepatic adenylate energy balance, and we expect that the depletion of ATP could contribute to an inhibition of liver glycolysis (56,57). AICAR also elevates plasma lactate in vivo (24,37), which may result from increased production from muscle (58) and impaired uptake in the liver (38).…”

AMP-activated protein kinase: an emerging drug target to regulate imbalances in lipid and carbohydrate metabolism to treat cardio-metabolic diseases

“…The effect of AICAR on mitochondrial respiration in intact hepatocytes at higher concentrations of AICAR (>100 M) was not mediated by AMPK, as this effect was also observed in hepatocytes isolated from AMPK ␣ 1 ␣ 2 liver-specifi c knockout mice ( 203 ). AMPK-independent effects of AICAR in the hepatic and muscle glucose uptake have been reported ( 54,288 ). AICAR gets metabolized to 5 amino imidazole-4-carboxamide ribonucleoside monophosphate (ZMP) as a result of uptake of AICAR (analog of adenosine) by the cells via an adenosine transport system, followed by conversion of AICAR to ZMP via adenosine kinase.…”

AMP-activated Protein Kinase Suppresses Matrix Metalloproteinase-9 Expression in Mouse Embryonic Fibroblasts

“…Furthermore, AICAR has been reported to inhibit cellular respiration by an AMPK-independent mechanism and decrease intracellular ATP (15)(16)(17)(18). These could account for the suppressive effects seen on MMP-9 expression in AMPK␣ Ϫ/Ϫ MEFs at the higher levels of AICAR (0.5 mM).…”