ever reaching an adequate level to induce glycogen synthesis. However, in CHO cells transfected with both GLUT1 and PTG, the lag phase was pronounced after even a brief exposure to 10 mM glucose. Thus, PTG, which stimulates glycogen synthesis by facilitating the interaction of the regulatory enzyme PP-1 with GS, GP and phosphorylase kinase, leads to greater glycogen synthesis during brief exposures to 10 mM glucose. According to our hypothesis, this elevates G-6-P levels for longer 8321748 after glucose removal and delays glucose clearance. In summary, these data are consistent with removal of glucose causing a switch from glycogen synthesis to glycogen breakdown, thereby increasing G-6-P which blocks the utilization of glucose by mitochondria-bound HK, in effect diverting G-6-P to glycolysis. 4 March 2011 | Volume 6 | Issue 3 | e17674 HK Localization and Glucose Fate Regulation of the metabolic fate of glucose by the subcellular distribution of HK 16632257 The observations so far indicate that HK1 overexpression affects catabolism, accelerating the t1/2 of glucose clearance, whereas HKII overexpression is anabolic, delaying the t1/2 by promoting glycogen synthesis, with subsequent inhibition of HK by glycogenolysis when extracellular glucose is removed. To determine how these metabolic roles are related to the subcellular localization of HKI and HKII, we studied the effects of extracellular glucose on subcellular HK distribution in CHO cells overexpressing HKI or HKII linked to YFP. Whether the cells were incubated in the presence or absence of glucose, MedChemExpress Oritavancin (diphosphate) HKI-YFP always remained associated with mitochondria, and addition or removal of glucose had no effect on its location . In contrast, the subcellular distribution of HKII-YFP was sensitive to extracellular glucose. In the presence of glucose, a large fraction of HKII was bound to mitochondria, and upon removal of extracellular glucose, HKII rapidly translocated to the cytosol with a time constant averaging 8.5 min . This effect was fully reversible, such that upon re-addition of glucose, HKII reassociated with mitochondria with a time constant of 15.5 min . These data support the hypothesis that HKI binds strongly to mitochondria, while the distribution of HKII between cytoplasm and mitochondria is labile, dynamically regulated by glucose availability. To evaluate the functional consequences of HKII redistribution on glucose metabolism, cells were subjected to a 20 to 30 min preincubation in the absence of glucose to maximize dissociation of HKII from mitochondria, and compared to cells with glucose present throughout. Preincubation in zero glucose prolonged the t1/2 of glucose clearance from 102611 s to 210619 s . This finding demonstrates that the subcellular distribution of HKII strongly influences its ability to promote anabolic use of glucose for glycogen synthesis. Since Akt facilitates HK interaction with mitochondria, we examined whether translocation of HKII from mitochondria to cytoplasm in response to zero glucose pre-incubation was suppressed by 
overexpression of constitutively activate Akt. With Akt overexpression, the mixed pattern of mitochondria-bound and cytosol-associated HKII-YFP did not change, and prolongation of the t1/2 of glucose clearance by zero glucose preincubation was abolished 107.069.5 s versus 210619 s with and without Akt). Thus, both glucose and Akt signaling promote the binding of HKII to mitochondria, favoring glucose catabolism over glycogen synthesis. March 2011 | V
