When oxygen becomes limiting, cells reduce mitochondrial respiration and increase ATP production through anaerobic fermentation of glucose. The Hypoxia Inducible Factors (HIFs) play a key role in this metabolic shift by regulating the transcription of key enzymes of glucose metabolism. Here we show that oxygen regulates the expression of the muscle glycogen synthase (GYS1). Hypoxic GYS1 induction requires HIF activity and a Hypoxia Response Element within its promoter. GYS1 gene induction correlated with a significant increase in glycogen synthase activity and glycogen accumulation in cells exposed to hypoxia. Significantly, knockdown of either HIF1α or GYS1 attenuated hypoxia-induced glycogen accumulation, while GYS1 overexpression was sufficient to mimic this effect. Altogether, these results indicate that GYS1 regulation by HIF plays a central role in the hypoxic accumulation of glycogen. Importantly, we found that hypoxia also upregulates the expression of UTP:glucose-1-phosphate urydylyltransferase (UGP2) and 1,4-α glucan branching enzyme (GBE1), two enzymes involved in the biosynthesis of glycogen. Therefore, hypoxia regulates almost all the enzymes involved in glycogen metabolism in a coordinated fashion, leading to its accumulation. Finally, we demonstrated that abrogation of glycogen synthesis, by knock-down of GYS1 expression, impairs hypoxic preconditioning, suggesting a physiological role for the glycogen accumulated during chronic hypoxia. In summary, our results uncover a novel effect of hypoxia on glucose metabolism, further supporting the central importance of metabolic reprogramming in the cellular adaptation to hypoxia.

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Phorbol esters, the archetypical (PKC) activators, induce apoptosis in androgen-sensitive LNCaP prostate cancer cells. In this study we evaluate the effect of a novel class of PKC ligands, the diacylglycerol (DAG)-lactones, as inducers of apoptosis in LNCaP cells. These unique ligands were designed using novel pharmacophore-and receptorguided approaches to achieve highly potent DAG surrogates. Two of these compounds, HK434 and HK654, induced apoptosis in LNCaP cells with much higher potency than oleoyl-acetyl-glycerol or phorbol 12,13-dibutyrate. Moreover, different PKC isozymes were found to mediate the apoptotic effect of phorbol 12-myristate 13-acetate (PMA) and HK654 in LNCaP cells. Using PKC inhibitors and dominant negative PKC isoforms, we found that both PKC␣ and PKC␦ mediated the apoptotic effect of PMA, whereas only PKC␣ was involved in the effect of the DAG-lactone. The PKC␣ selectivity of HK654 in LNCaP cells contrasts with similar potencies in vitro for binding and activation of PKC␣ and PKC␦. Consistent with the differences in isoform dependence in intact cells, PMA and HK654 show marked differences in their abilities to translocate PKC isozymes. Both PMA and HK654 induce a marked redistribution of PKC␣ to the plasma membrane. On the other hand, unlike PMA, HK654 translocates PKC␦ predominantly to the nuclear membrane. Thus, DAG-lactones have a unique profile of activation of PKC isozymes for inducing apoptosis in LNCaP cells and represent the first example of a selective activator of a classical PKC in cellular models. An attractive hypothesis is that selective activation of PKC isozymes by pharmacological agents in cells can be achieved by differential intracellular targeting of each PKC.The phorbol esters and related diterpenes are natural compounds that have been for many years the preferred pharmacological tools for studying protein kinase C (PKC), 1 a key family of kinases implicated in growth factor-and G-proteincoupled receptor signaling. These compounds mimic the action of the lipid second messenger diacylglycerol (DAG), a relatively simple and highly flexible molecule generated by cellular phospholipases. The higher potency of phorbol esters and their greater stability compared with the second messenger DAG makes these agents the preferred activators of PKC (1, 2). Phorbol esters regulate a variety of cellular functions, including cell cycle progression, differentiation, cytoskeleton remodeling, and malignant transformation. Although phorbol esters promote mitogenesis in several cell types, accumulating data indicate that activation of PKC leads to inhibition of cell growth in many cells (3-6). Interestingly, phorbol esters induce apoptosis in several cell lines, including thymocytes, breast cancer cells, and prostate cancer cells (7-12).The heterogeneity of effects of the phorbol esters is related to the presence of multiple phorbol ester/DAG receptors, including PKC isozymes and "nonkinase" PKC receptors. The PKC family comprises at least 10 related kinases with differential expression, ...

The members of the chimaerin family of Rac-GTPase-activating proteins possess a single C1 domain with high homology to those present in protein kinase C (PKC) isozymes. This domain in PKCs is involved in phorbol ester and diacylglycerol (DAG) binding. We previously have demonstrated that one of the chimaerin isoforms, ␤2-chimaerin, binds phorbol esters with high affinity. In this study we analyzed the properties of ␤2-chimaerin as a DAG receptor by using a series of conformationally constrained cyclic DAG analogues (DAG lactones) as probes. We identified analogs that bind to ␤2-chimaerin with more than 100-fold higher affinity than 1-oleoyl-2-acetylglycerol. The potencies of these analogs approach those of the potent phorbol ester tumor promoters. The different DAG lactones show some selectivity for this novel receptor compared with PKC␣. Cellular studies revealed that these DAG analogs induce translocation of ␤2-chimaerin from cytosolic (soluble) to particulate fractions. Using green fluorescent protein-fusion proteins for ␤2-chimaerin we determined that this novel receptor translocates to the perinuclear region after treatment with DAG lactones. Binding and translocation were prevented by mutation of the conserved Cys-246 in the C1 domain. The structural homology between the C1 domain of ␤2-chimaerin and the C1b domain of PKC␦ also was confirmed by modeling analysis. Our results demonstrate that ␤2-chimaerin is a high affinity receptor for DAG through binding to its C1 domain and supports the emerging concept that multiple pathways transduce signaling through DAG and the phorbol esters. Signaling in response to the second messenger diacylgycerol (DAG) is thought to proceed through the activation of protein kinase C (PKC) isozymes (1, 2). Binding of this lipid second messenger and its related analogs, the phorbol esters, occurs at the C1 domains (also called cysteine-rich regions or zinc fingers) present in the classical PKCs (PKC␣, ␤I, ␤II, and ␥) and novel PKCs (PKC␦, , , and ). This 50-to 51-aa domain, which is present in tandem in these PKC isozymes, possesses the motif HX 12 CX 2 CX n CX 2 CX 4 HX 2 CX 7 C, where H is histidine, C is cysteine, X is any other amino acid, and n is 13-14 (3-6). The phorbol ester receptor family has expanded with the discovery of the chimaerins. Unlike PKCs, the chimaerins do not possess a functional kinase domain but they are GTPase-activating proteins for Rac, a small GTP binding protein of the Ras superfamily (7). Four chimaerin isoforms (␣1-or n-, ␣2-, ␤1-, and ␤2-chimaerin) have been identified to date, all of them possessing a single C1 domain with approximately 40% homology to those present in PKCs (7-10). It is therefore predictable that the biological responses of the phorbol esters and those mediated by DAG signaling could involve the activation of PKC-independent pathways.We previously have reported that ␣1-and ␤2-chimaerin are indeed high affinity receptors for the phorbol esters and also for the bryostatins, macrocyclic lactones with antitumor properties (11,12). ...